scholarly journals Nile Red Quantifier: a novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy

2017 ◽  
Vol 58 (11) ◽  
pp. 2210-2219 ◽  
Author(s):  
Johan G. Schnitzler ◽  
Sophie J. Bernelot Moens ◽  
Feiko Tiessens ◽  
Guido J. Bakker ◽  
Geesje M. Dallinga-Thie ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Lipid Accumulation ◽  
Nile Red ◽  
Quantitative Tool

scholarly journals Nile Red quantifier: A novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy

2017 ◽  
Vol 263 ◽  
pp. e146
Author(s):  
Jan Schnitzler ◽  
Feiko Tiessens ◽  
Sophie Bernelot Moens ◽  
Geesje Dallinga-Thie ◽  
Albert Groen ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Lipid Accumulation ◽  
Nile Red ◽  
Quantitative Tool

scholarly journals Honokiol Attenuates Lipotoxicity in Hepatocytes via Activating SIRT3-AMPK Mediated Lipophagy

2021 ◽  
Author(s):  
Zhang Tian ◽  
Jingxin Liu ◽  
Jianzhong Zhu ◽  
Rongsong Li ◽  
Ligen Lin
Keyword(s):  
Lipid Accumulation ◽  
Energy Homeostasis ◽  
Nile Red ◽  
Acid Mixture ◽  
Amino Acid Residues ◽  
Alcoholic Fatty Liver ◽  
Docking Analysis ◽  
Chain Acyl ◽  
Intracellular Lipid Accumulation ◽  
Amp Activated Protein Kinase

Abstract Background: Non-alcoholic fatty liver disease (NAFLD) is characterized by ectopic accumulation of triglycerides in the liver. Emerging evidence has demonstrated that lipophagy regulates lipid mobilization and energy homeostasis in liver. Sirtuin 3 (SIRT3), a mitochondrial NAD+-dependent deacetylase, modulates the activities of several substrates involving in autophagy and energy metabolism. Honokiol (HK) is a natural lignan from the plants of Magnolia genus that exhibits potent liver protective property. Methods: AML12 was challenged with 500 μM palmitic acid and 250 μM oleic acid mixture solution to induce lipotoxicity. The expression of autophagy-related and AMP-activated protein kinase (AMPK) pathway proteins was evaluated by Western blotting and immunofluorescence staining. Intracellular lipid accumulation was validated by Nile red staining. Molecular docking analysis was performed on AutoDock 4.2.Results: HK (5 and 10 μM) was found to attenuate lipid accumulation through promoting SIRT3-AMPK-mediated autophagy, mainly on lipid droplets. HK had hydrophobic interaction with amino acid residues (PHE294, GLU323 and VAL324) and NAD+. Moreover, HK improved mitochondrial function to enhance lipolysis, through decreasing the acetylated long-chain acyl-CoA dehydrogenase level. Conclusions: These results suggest that HK could ameliorate lipotoxicity in hepatocytes by activating SIRT3-AMPK-lipophagy axis, which might be a potential therapeutic agent against NAFLD.

scholarly journals Initial study of lipid accumulation in green algal Haematococcus pluvialis Flotow cultured in liquid Bold’s Basal medium aerated

2019 ◽  
Vol 3 (3) ◽  
pp. 144-149
Author(s):  
Nguyen Tran Dong Phuong ◽  
Le Huyen Ai Thuy ◽  
Bui Trang Viet
Keyword(s):  
Lipid Accumulation ◽  
Fatty Acid Biosynthesis ◽  
Haematococcus Pluvialis ◽  
Acyl Carrier Protein ◽  
Nile Red ◽  
Basal Medium ◽  
Initial Study ◽  
Rt Pcr ◽  
Green Algal ◽  
High Lipid

The fresh green algal Haematococcus pluvialis Flotow was proved to be the starting material for the production of biofuel, high lipid content along with astaxanthin, a high value colorant. In this study, lipid accumulation in H. pluvialis cultured in liquid Bold’s Basal medium aerated was investigated for a period of 12 weeks. Lipid accumulation was evaluated through the expression of two genes: BC (biotin carboxylase, initial gene) and FATA (acyl-acyl carrier protein thioesterase, end gene) in the process of fatty acid biosynthesis with Real-time RT-PCR, lipid determination by Nile Red and biodiesel quantifying by transesterification. The results showed that the expression of two BC and FATA genes was recorded at all weeks of culture. However, the expression of BC and FATA genes increased gradually from the week 9 (1.3, 4.1, respectively) to week 11 (1.7, 30.9, respectively). Meanwhile, yellow fluorescence in the microalgal cells showed that lipid appeared from week 6 to week 12. The obtained biodiesel increased slowly from week 8 (0.036 mg/mL) to week 12 (0.041 mg/mL). At week 11, the expression values of both BC gene (1.7) and FATA gene (30.9) were maximized, leading to the highest biodiesel content at the week 12.

Feeding induces lipid accumulation and increased Na+ transport in in vitro Necturus antrum

1990 ◽  
Vol 259 (6) ◽  
pp. G998-G1009
Author(s):  
M. J. Rutten ◽  
C. D. Moore ◽  
R. Delcore ◽  
L. Y. Cheung
Keyword(s):  
Electron Microscopy ◽  
Lipid Accumulation ◽  
Corn Oil ◽  
Short Circuit ◽  
Nile Red ◽  
Transepithelial Transport ◽  
Na Transport ◽  
Ussing Chambers ◽  
Lipid Granules

We investigated the effects of feeding on lipid accumulation and transepithelial transport using in vitro Necturus gastric antral mucosae. Antra from fed Necturi were examined for lipid accumulation using light, fluorescence, histochemical, and electron microscopy. Ussing chambers were used for measurement of potential difference (PD), transepithelial resistance (Rt), short-circuit current (Isc), and unidirectional fluxes of 22Na+ and [3H]mannitol. Light microscopy of antra from 2-day postfed animals showed many intracellular lipid granules in surface mucous epithelial cells. These granules could be distinguished from other intracellular organelles by their high affinity for osmium and the lipid fluorescent probe Nile red. Glycoprotein cytochemical staining showed these granules to be distinct from the epithelial cell mucous granules. Electron microscopy showed the lipid granules to be part of a membranous reticular network. Two-day postfed animals also had a approximately 3.5-fold increase in amiloride-sensitive Isc and PD, a decrease in Rt, and an increased luminal-to-serosal Na+ fluxes. Transepithelial [3H]mannitol fluxes were low and remained unchanged in both fasted and 2-day postfed animals. After 2 days of feeding, the PD and Isc began to decrease followed by a secondary increase in Rt. Feeding Necturi a corn oil diet did not induce the appearance of either cellular lipid or alterations in Isc but produced a transient increase in Rt. Our data show that feeding (goldfish) to Necturi causes an increase in both lipid accumulation and amiloride-sensitive Na+ transport in gastric antral cells.

109 EFFECTS OF LIPOLYTIC AGENTS FORSKOLIN, EPINEPHRINE AND CAFFEINE ON EMBRYONIC DEVELOPMENT AND LIPID CONTENT OF BOVINE EMBRYOS PRODUCED IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 154 ◽  
Author(s):  
M. Barcelo-Fimbres ◽  
G. E. Seidel
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Embryonic Development ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Nile Red ◽  
Essential Amino Acids

The objective of this experiment was to evaluate lipid accumulation and embryonic development of bovine morulae treated with different chemicals. A total of 2619 slaughterhouse oocytes from heifers and mature cows were matured in CDM medium (similar to SOF) plus 0.5% fatty acid-free BSA and hormones (M-CDM) for 23 h at 38.5°C in 5% CO2 in air. Frozen–thawed sperm were centrifuged through a Percoll gradient and co-cultured with matured oocytes for 18 h in F-CDM (CDM+heparin). Zygotes were cultured at 38.5°C in 5% CO2/5% O2/90% N2 in CDM-1 with nonessential amino acids, 10 μm EDTA, 0.5% fatty acid free BSA, and 0.5 mm fructose. After 60 h, resulting 8-cell embryos were cultured 120 h in CDM-2 (CDM-1+essential amino acids and 2 mm fructose). A factorial design was used with 7 treatments, 2 ovary sources (cows v. heifers), and 3 bulls (A, B and C) replicated twice for each bull (6 replicates). At Day 2.5 embryo cleavage and 8-cell rates were evaluated, and on Day 6 a total of 755 morulae were randomly assigned to the 7 treatments (control, 2 and 8 mm caffeine, 1 and 4 μm epinephrine, and 10 and 40 μm forskolin). To quantify lipid accumulation, Day 7 blastocysts were fixed and stained with 1 μg mL–1 Nile red dye, after which a digital photograph of the equatorial part of the embryo (including the inner cell mass) was taken at 200×, and fluorescence intensity was measured with Image Pro software from 0 to 255 shades for each pixel (0 = no lipids; 255 = greatest lipid accumulation), as previously reported (Biol. Reprod. 2007 (Suppl. 1), 87–88). Data were analyzed by ANOVA. No differences in cleavage rates (75 v. 68 ± 3.6%) or eight cell rates (61 ± v. 57 ± 2.8%) were found for heifer v. cow oocytes (P > 0.1); however, blastocyst rates per oocyte and per 8-cell embryo were greater for cows than heifers (20 v. 10 ± 2.1%, and 68 v. 35 ± 3.8%, respectively; P < 0.05). Treatments: 2 and 8 mm caffeine produced fewer blastocysts per morula than 1 and 4 μm epinephrine, 10 and 40 μm forskolin and the control (39, 5 v. 54, 49, 48, 54 and 52 ± 5.8%; respectively) (P < 0.01). More lipid content was found in whole embryos and trophoblast of heifer-derived than cow blastocysts (P < 0.05), and forskolin resulted in less lipid content than control, caffeine- and epinephrine-treated morulae in whole embryos, embryonic mass and trophoblasts (P < 0.05; Table 1). In conclusion, mature cows were a better source of oocytes than feedlot heifers for embryonic development. High doses of caffeine were detrimental to embryos, and the addition of the lypolitic agent forskolin reduced lipid content relative to control, caffeine and epinephrine-treated embryos. Table 1.Main effect treatment means of lipid content (arbitrary fluorescence units)

89 EFFECT OF MEDIA, METABOLIC REGULATORS, AND STAGE OF DEVELOPMENT ON LIPID CONTENT AND MITOCHONDRIAL POLARITY OF IN VITRO-PRODUCED HOLSTEIN EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 174
Author(s):  
M. A. Roberts ◽  
L. F. Campos-Chillon ◽  
M. Barceló-Fimbres ◽  
J. L. Altermatt
Keyword(s):  
Fluorescence Intensity ◽  
Lipid Content ◽  
Lipid Accumulation ◽  
Metabolic Regulation ◽  
Nile Red ◽  
Bovine Embryo ◽  
Stage Of Development ◽  
Mitotracker Red ◽  
Metabolic Regulators

Current bovine embryo culture methods result in accumulation of lipids and reactive oxygen species, possibly due to sub-optimal metabolic regulation. These effects decrease the cryopreservation survival and implantation potential of in vitro-produced (IVP) embryos. Forskolin has been shown to decrease lipid accumulation, and vitamin K2 (Vit K2) is thought to decrease oxidative stress from in vitro conditions. The aims of this study were (1) to assess lipid content of embryos cultured with or without forskolin and Vit K2 in both continuous and sequential SOF-based medium, and (2) to examine individual and combined effects of forskolin and Vit K2 on mitochondrial polarity. For Experiment 1, a 2 × 2 × 2 factorial design was used to compare culture systems (continuous v. 3-step sequential), additives (no additive v. Vit K2 (0.5 mM at Day 3) plus forskolin (10 µM at Day 5), and blastocyst stage [6 (early) v. 7 (late)] on overall lipid content. For Experiment 2, mitochondrial polarity of stage 7 blastocysts was analysed from the following groups: no additive, Vit K2 (0.5 mM at Day 3), forskolin (10 µM at Day 5), and Vit K2 plus forskolin. IVP embryos (n = 199, Experiment 1; n = 45, Experiment 2) were produced by standard procedures and cultured at 38.5°C in 5% O2, 5% CO2, and 90% N2. For Experiment 1, embryos were stained with 1 μg mL–1 Nile Red, and two images per embryo were taken along the equatorial plane at 40× magnification. For Experiment 2, embryos were stained with 300 nM MitoTracker Red CMX-Rosamine, and 10 images per embryo were acquired by confocal microscopy with a 5-μm step size at 40× magnification. For both experiments, fluorescence intensity (FI) of each image was measured by Image PRO software with embryo controlled for and background fluorescence corrected. Data (Table 1) were analysed by ANOVA and means were compared by Tukey’s HSD. In Experiment 1, embryos cultured with forskolin and Vit K2 showed decreased lipid content in both the early and late stage (P < 0.05), with no effect from culture system (P > 0.05). In Experiment 2, forskolin and Vit K2 individually increased mitochondrial polarity (P < 0.05), but had no combined effect (P > 0.05). In conclusion, these data suggest that while a combination of forskolin and Vit K2 as media additives reduces lipid accumulation, the interaction between these metabolic regulators may negate their individual effects on mitochondrial polarity. Table 1.Fluorescence intensity of Nile Red and MitoTracker Red dyes between treatment groups

Monitoring cell-specific neutral lipid accumulation in Phaeodactylum tricornutum (Bacillariophyceae) with Nile Red staining - a new method for FlowCAM

2017 ◽  
Vol 53 (2) ◽  
pp. 396-404 ◽  
Author(s):  
Katariina Natunen ◽  
Jukka Seppälä ◽  
Riikka-Juulia Koivula ◽  
Jukka Pellinen
Keyword(s):  
Neutral Lipid ◽  
Lipid Accumulation ◽  
Phaeodactylum Tricornutum ◽  
Nile Red ◽  
New Method ◽  
Nile Red Staining

scholarly journals Nile Red and 2-NBDG Are Incompatible for the Simultaneous Detection of Lipid and Glucose Accumulation

2016 ◽  
Vol 2016 ◽  
pp. 1-8 ◽  
Author(s):  
Andrew M. Hogan ◽  
Viswanathan Swaminathan ◽  
Nikitha K. Pallegar ◽  
Sherri L. Christian
Keyword(s):  
Glucose Uptake ◽  
Lipid Accumulation ◽  
Mammalian Cells ◽  
Simultaneous Detection ◽  
Nile Red ◽  
Lipid Synthesis ◽  
Cell Types ◽  
Fluorescent Intensity ◽  
Fine Control ◽  
Glucose Accumulation

Glucose is the universal energy source and a critical substrate for lipid synthesis in mammalian cells. Analysis of both glucose and lipid in cells is important for the understanding of the regulation of lipid synthesis in many cell types, but especially adipocytes, the major storage cell for fat in mammals. The fluorescent 7-nitrobenz-2-oxa-1,3-diazole (NBD) derivative of glucose, 2-NBDG, is used to monitor glucose uptake and the lipid-selective fluorophore Nile red is used to monitor lipid accumulation. Previous reports have used NBD-based fluorophores and Nile red simultaneously despite the possibility of spectral overlap. In this study, we determined if these fluorophores were experimentally compatible in preadipocytes and adipocytes stained with 2-NBDG and Nile red separately or costained. We found that Nile red is detectable in the wavelengths necessary to excite and detect 2-NBDG. This interference was further increased by the solvatochromic effect of lipid-localized Nile red. In addition, we found a synergistic increase in fluorescent intensity when both fluorophores were present. Unfortunately, even fine control of the excitation or emission wavelengths did not identify wavelengths suitable for selective detection when cells were costained. Therefore, 2-NBDG and Nile red cannot be used simultaneously—but can likely be used sequentially—to assess glucose uptake and lipid accumulation in lipid-laden cells.

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